The effect of Brownian motion of fluorescent probes on measuring nanoscale distances by Forster resonance energy transfer
The effect of Brownian motion of fluorescent probes on measuring nanoscale distances by Forster resonance energy transfer
复制标题
DOI：
10.1063/1.3598109
复制
发表时间：
2011-06-14
影响因子：
4.4
通讯作者：
Gradinaru, Claudiu C.
中科院分区：
文献类型：
--
作者：
Badali, Daniel;Gradinaru, Claudiu C.
福斯特共振能量转移（FRET）是一种强大的光学技术，以确定分子内的距离。然而，染料旋转运动和连接体柔性使测量的能量转移效率与染料的锚定点之间的距离之间的关系复杂化。在这项研究中，我们提出了一个简单的模型，描述了链接器和染料的动力学扩散在一个球。单对能量转移被处理在弱激发极限，光子统计和支架的灵活性被忽略，并考虑不同的时间平均制度。尽管近似，我们的模型提供了新的见解，实验设计和结果解释单分子FRET。Monte Carlo模拟产生了染料间距离、偶极取向因子kappa（2）和转移效率E的分布，这些分布与独立推导的理论函数完全一致。与通常的看法相反，我们的数据表明，较长的连接体实际上会限制染料偶极的运动，从而使kappa（2）的各向同性2/3近似变差。它也被发现，染料连接系统的热运动引起快速和大的效率波动，如所示的模拟FRET时间轨迹上的微秒的时间尺度上分仓。单分子FRET测量的基本分辨率极限出现在1-10 μ s左右，这应该被考虑用于解释在这样快的时间尺度上记录的数据。(C)2011年美国物理学会。[doi：10.1063/1.3598109]
Forster resonance energy transfer (FRET) is a powerful optical technique to determine intramolecular distances. However, the dye rotational motion and the linker flexibility complicate the relationship between the measured energy transfer efficiency and the distance between the anchoring points of the dyes. In this study, we present a simple model that describes the linker and dye dynamics as diffusion on a sphere. Single-pair energy transfer was treated in the weak excitation limit, photon statistics and scaffold flexibility were ignored, and different time-averaging regimes were considered. Despite the approximations, our model provides new insights for experimental designs and results interpretation in single-molecule FRET. Monte Carlo simulations produced distributions of the inter-dye distance, the dipole orientation factor, kappa(2), and the transfer efficiency, E, which were in perfect agreement with independently derived theoretical functions. Contrary to common perceptions, our data show that longer linkers will actually restrict the motion of dye dipoles and hence worsen the isotropic 2/3 approximation of kappa(2). It is also found that the thermal motions of the dye-linker system cause fast and large efficiency fluctuations, as shown by the simulated FRET time-trajectories binned on a microsecond time scale. A fundamental resolution limit of single-molecule FRET measurements emerges around 1-10 mu s, which should be considered for the interpretation of data recorded on such fast time scales. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3598109]